18β-甘草次酸对金黄色葡萄球菌肺炎的保护作用及其机制研究

发布时间：2018-05-29 11:07

本文选题：18β-甘草次酸 + 金黄色葡萄球菌　；参考：《浙江理工大学》2017年硕士论文

【摘要】：目的:建立小鼠金黄色葡萄球菌(SA)肺炎动物模型,研究18β-甘草次酸(18β-GA)对SA肺炎的保护作用,并进一步探讨其分子机制。方法:6-8周龄C57/BL6雌鼠随机分为3组,对照组:小鼠气道滴注磷酸盐缓冲液(PBS);SA模型组:小鼠气道滴注SA(1×108 CFU);18β-GA干预组:小鼠在SA气道滴注前2小时,予腹腔注射18β-GA(50mg/kg)。观察3组小鼠生存率变化,检测各组小鼠肺组织和肝组织病理改变,肺泡灌洗液(BALF)中性粒细胞计数,用ELISA检测BALF中炎症因子表达,RT-q PCR测定肺组织炎症因子表达,CFU测定肺组织细菌负荷,Western Blot测定肺组织高迁移率蛋白1(HMGB1)表达。另一组实验观察HMGB1抗体对SA肺炎小鼠生存率的影响。用18β-GA(20μM)预处理巨噬细胞RAW264.7和肝细胞Hep G2,再加入SA刺激。ELISA检测巨噬细胞RAW264.7上清液中炎症因子的表达,Western Blot检测P38/p-P38MAPK,JNK/p-JNK MAPK,ERK/p-ERK MAPK和P65/p-P65 NF-κB相关信号蛋白的表达。检测肝细胞Hep G2上清的谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性。结果:体内实验发现:预先腹腔注射18β-GA对SA肺炎及继发性肝损伤具有保护作用。与SA模型组比较,18β-GA干预可以减轻SA引起的肺组织和肝组织病理损伤,18β-GA预处理组小鼠BALF中性粒细胞计数显著降低(P0.05);炎症因子(肿瘤坏死因子TNF-α,白细胞介素IL-1β,IL-6)下调(P0.05),肺组织细菌载量降低(P0.05)。18β-GA可显著抑制HMGB1在感染小鼠肺组织中的表达,腹腔注射HMGB1抗体可以显著提高SA肺炎小鼠的生存率(P0.05)。体外实验提示:18β-GA通过抑制巨噬细胞P65 NF-κB蛋白磷酸化,显著降低炎症因子表达(P0.05),同时可以直接抑制SA生长;18β-GA也可降低SA诱导的肝细胞ALT表达(P0.05)。结论:18β-GA对SA肺炎和继发性肝损伤有保护作用。18β-GA通过减少炎症介质产生和降低损伤模式分子HMGB1表达,发挥保护作用。
[Abstract]:Objective: to establish an animal model of SAA pneumonia in mice, to study the protective effect of 18 尾 -glycyrrhetinic acid (18 尾 -GAA) on SA pneumonia and to explore its molecular mechanism. Methods C57/BL6 female mice aged 6-8 weeks were randomly divided into three groups. The control group was treated with SA(1 脳 108CFU 18 尾 -GA. The mice were injected intraperitoneally with 18 尾 -GAN 50 mg / kg before the infusion of SA. The survival rate of the three groups was observed, the pathological changes of lung and liver tissues and the neutrophil count in alveolar lavage fluid (BALF) of each group were detected. Expression of inflammatory factors in BALF was detected by ELISA RT-Q PCR. CFU was used to detect the expression of inflammatory factors in lung tissue. Western Blot was used to detect the expression of high mobility protein 1 (HMGB1) in lung tissue. In another group, the effect of HMGB1 antibody on survival rate of SA pneumonia mice was observed. Macrophages RAW264.7 and hepatocyte Hep G2 were pretreated with 18 尾 -GAN 20 渭 M, and then SA stimulated. Elisa was added to detect the expression of inflammatory factors in the supernatant of macrophage RAW264.7. Western Blot was used to detect the expression of P38 / p-P38 MAPK / JNKP / p-JNK MAPK / ERKP / ERK MAPK and P65/p-P65 NF- 魏 B related signal proteins. The activities of alanine aminotransferase (alt) and aspartate aminotransferase (AST) in the supernatant of Hep G2 were measured. Results: in vivo, 18 尾-GA had protective effect on SA pneumonia and secondary liver injury. Compared with the SA model group, the intervention of TNF- 18 尾 -GA could attenuate the pathological injury of lung and liver tissue induced by SA. The neutrophil count of BALF in the preconditioning group of 18 尾 -GA significantly decreased P0.05, and the inflammatory factor (TNF- 伪, interleukin IL-1 尾 -IL-6) down-regulated P0.05G. The decrease of bacterial load in lung tissue could significantly inhibit the expression of HMGB1 in lung tissue of infected mice. Intraperitoneal injection of HMGB1 antibody could significantly improve the survival rate of SA pneumonia mice (P 0.05). The in vitro experiments showed that the expression of P0.05A was significantly decreased by inhibiting the phosphorylation of p65 NF- 魏 B protein in macrophages, and that the growth of SA was inhibited directly by 18 尾 -GA, and the expression of ALT in hepatocytes induced by SA was also decreased. Conclusion.18 尾 -GA has a protective effect on SA pneumonia and secondary liver injury. 18 尾 -GA plays a protective role by reducing the production of inflammatory mediators and reducing the expression of HMGB1.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.1

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